Globally, protozoan pathogens are an increasingly important cause of reported disease outbreaks, with the majority of documented outbreaks between 2004 and 2010 reported in Australia. While the microbiological contamination of roof-harvested rainwater (RHRW) has been well studied, limited information is available regarding contamination with protozoan pathogens. In this study, rainwater (n = 134) and possum fecal samples (n = 20) were screened for the presence of several protozoan pathogens, including Blastocystis spp., Cryptosporidium spp., Giardia spp., Dientamoeba fragilis, and Entamoeba histolytica using the multiplex real-time polymerase chain reaction. While Cryptosporidium spp. was only detected in two possum fecal samples (10%) and Giardia spp. was only detected in three RHRW samples (2.23%, n = 134), Blastocystis spp. was detected in both possum feces (25%) and RHRW (5.22%) samples. Dientamoeba fragilis and Entamoeba histolytica were not detected in any samples. These findings highlight protozoan pathogens as a potentially important area of focus for rainwater quality assessment. Furthermore, while possums are suggested as a potential source of Blastocystis spp. in RHRW, sources of this pathogen in RHRW warrant further investigation.